We describe a procedure for simplified, simultaneous one-step staining in 10 min for
DNA and cell and tissue
proteins using a newly developed staining
solution containing 0.03%
hematoporphyrin (HP) with 0.001%
DAPI [or with Hoeschst 33342 (HO)]. These HP/
DAPI or HP/HO solutions were especially developed to facilitate a trial of automated
cancer cell screening on sputum samples using flow cytometry. Under UV light (365 nm) with fluorescence microscopy, HP/
DAPI-stained cells showed red fluorescence (max. 670 nm) of cytoplasm and simultaneous blue fluorescence (max. 470 nm) of nuclei. The distance between the maximum peak of fluorescence spectra of
DNA and that of
protein was as large as 200 nm, and there was no detectable overlapping of each spectrum at the photometric filter range, which provided accurate measurement of
DNA and
protein. On flow cytometry, a single UV beam (370 nm) from the
argon laser was used for excitation of both
dyes. Measurement of
DNA was done using a 470-nm bandpass filter and of
protein using a 640-nm longpass (or 670-nm bandpass) filter. Reflecting the undetectable overlapping of the fluorescence spectra of
protein and
DNA, normal diploid cells in sputum revealed horizontal distributions along the 2C level on the dot-plot display of flow cytometry, which made sorting of abnormal hyperdiploid cells and
cancer cells easier.